The negative-stranded RNA viruses comprise some of the most serious human pathogens in terms of both mortality and morbidity. The purpose of the research described here is to study the mechanism of replication of the prototypical negative-stranded rhabdovirus, vesicular stomatitis virus (VSV) and the unusual human paramyxovirus respiratory syncytial (RS) virus. This research is designed to understand at the molecular level the factors relevant in controlling expression of the information on the genome of these viruses. We will investigate the relative roles of two factors: (i) individual viral proteins and (ii) primary RNA sequence, in controlling the decision to express the genome by transcription to yield discrete mRNAs or by replication to provide a complete copy of the genome. The requirement for individual VSV specific proteins in replication is being dissected using an in vitro replication system in which replication is a function of ongoing protein synthesis programmed by addition of individual mRNAs. A method to generate specific mutations in RNA genomes at the cDNA level and to recover precise RNA transcripts of that altered cDNA is proposed. This method will be used to evaluate the role of sequence in controlling RNA synthetic events and to investigate other structure-function relationships. The specific aims of this grant are as follows: 1) Determine whether proteins other than N are involved in regulation the transition from transcription to replication. 2) Develop a system to create defined mutations in negative-strand RNA viruses. 3) Determine the N protein encapsidation site using deletion and site-specific mutagenesis. 4) Analyze the role of sequence at the leader:N and intercistronic junctions in controlling the decision to transcribe or replicate. 5) Analyze RS virus RNA transcription and the role of RS virus specific nonstructural proteins in RNA synthesis. 6) Develop an in vitro RNA synthesis system for respiration syncytial virus transcription and replication.